Eukaryotic translation initiation factors control several steps in the assembly of mRNA with 40S ribosomal subunits at the start of protein synthesis. Two of these factors, elF2 and elF3, are almost universally required for cap-dependent and cap-independent initiation. Eukaryotic initiation factor elF2 is a heterotrimer responsible for bringing initiator tRNA to ribosome-mRNA assemblies, and represents one of the key targets of negative translation regulation by kinases. Initiation factor elF3 includes at least twelve non-identical subunits and is required for translation of most m7G-cap- and IRES-driven mRNAs. The internal ribosome entry site (IRES) of hepatitis C virus (HCV) utilizes both elF2 and elF3 during translation initiation, and a direct contact between the IRES and elF3 is essential for efficient viral translation. This proposal aims to understand the biochemical and structural basis for the activities of these factors. We will develop methods for large-scale purification of these factors and will analyze their composition both alone and within functional ribosomal complexes defined by the HCV IRES using quantitative mass spectrometry. We will determine the role of elF3 in HCV IRES-mediated translation initiation by isolating and analyzing translation complexes that bind the wild type and mutant forms of the IRES in vitro and in vivo. Finally, we plan to determine molecular structures of elF2 and elF3 using both X-ray crystallography and electron microscopy, providing a structural framework for interpreting interactions that occur during both IRES-mediated and cap-dependent translation. Direct collaboration and interactions with the Gate, Harris, Hershey and Sarnow laboratories, as well as the proposed Mass Spectrometry Core Laboratory to be directed by Prof. Leary, is an integral part of these experiments.